Centrifugal filters may be used to separate biological substances such as an antibody enzyme, nucleic acid and protein for the purpose of concentration, desalting, purification, and fractionation. These devices are most commonly used in centrifugal-separator instruments, which may consist of a fixed-angle-rotor configuration or a swing- or variable-angle-rotor configuration. The speed of the filtering process and the recovery of retentate sample are highly valued by customers. Sample recovery values higher than 85% are usually obtained by removing the membrane capsule (sample holder) and reverse spinning it in a receiver tube.
Such devices are typically used to concentrate urine, serum, plasma and cerebrospinal fluid. For example, the measurement of specific proteins in urine can be important for the diagnosis and management of various disease states, yet the content of these proteins in urine is often too small to be detected without first concentrating the proteins. Conventional devices generally include a housing having a sample reservoir, a filter sealed in the housing so that the sample must past through the filter when subject to a driving force (such as centrifugation), and a collection chamber for collecting the concentrated sample.
There is a class of protein purification protocols that use antigen-protein affinity to separate proteins of interest from a mixed sample such as a cell lysate or serum. Such protocols often use small beads that are conjugated with antibodies such that they bind to specific proteins from the sample. Once the proteins are effectively bound to the beads, there is a need to extract and collect the proteins (elution) from the beads for downstream analysis, assay development, etc. Exemplary downstream analysis techniques include 2D gel electrophoresis and mass spectrometry.
There are a number of processing steps that are needed in the workflow. These can include equilibrating the beads with neutral buffer prior to binding, washing the beads after binding to remove unbound contaminates, eluting the proteins of interest, exchanging the buffer from the eluted proteins, concentrating the final diluted sample, and finally recovering the purified proteins sample. For affinity purification and immunoprecipitation protocols, the proteins bound to the beads are the proteins of interest. For depletion protocols, the unbound fraction (proteins not bound to the beads) is the sample of interest.
Beads used in these purification methods are magnetic or non-magnetic. One of the most common non-magnetic beads is agarose. Magnetic beads such as PureProteome protein A & G, PureProteome albumin and PureProteome albumin and IgG for albumen and IgG depletion from serum, Magna ChIP protein A beads for chromatic immuniprecipitation, and PureProteome Nickel magnetic beads for His-tagged recombinant purification, are commercially available from EMD Millipore.
When working with magnetic beads, current manual methods rely on the use of pipettes to move liquids to and from the sample tube (buffers, etc.) and to move the sample from one device to another. Magnets are used to hold the beads to the side of the sample tube so that the user can pipette out the buffers without disturbing the beads. There are about 8 pipette steps per sample in a typical bind/wash/elute workflow.
For optimal protein binding with the beads, incubation is required with these methods. The device containing the beads and the sample are usually turned in an end-over-end mixer, or placed in a shaker (e.g., vortexor) for 10-30 minutes. When new buffers are added, such as wash and elution buffers, the user will vortex the device for a minute or so to mix and wash.
The washing and eluting steps need to be repeated multiple times in order to be effective. For example, standard protocol is to add wash buffer to the sample vial, vortex (mix) for a minute or so, remove the buffer and repeat two or more times. With magnetic beads, the bind/wash/elute procedure takes about 45 minutes.
An alternative to magnetic beads is agarose beads. One commercially available device that uses agarose beads includes a tube with an open bottom and a porous frit positioned over the open bottom. Instead of using pipettes to remove fluids from the sample tube, a bench top centrifuge is used to drive the fluids through the frit and into a collection tube—typically a 4 mL or 15 mL tube. The frit pore size is chosen to retain the beads while allowing buffers and proteins to pass through.
Depending on the size of the spin column used, the workflow can be cumbersome and time consuming compared to methods that use magnetic beads. A bench top centrifuge is typically a shared piece of equipment located at a common location; unlike microcentrifuges that each user may have setup at their work area.
This process requires 16 pipetting steps per sample and takes about 1 hour to complete.
For both magnetic and agarose workflows, downstream steps may include exchanging the carrier buffer and concentrating a diluted sample. In cases where buffer exchange of the sample is desired, perhaps to remove the eluent like imidazole, the sample is typically transferred to a dialyzing membrane tube with clamps or the like, which is then placed inside a tank of exchange buffer for up to 24 hours as the buffer is exchanged gradually by way of diffusion.
Where buffer exchange and concentration is desired, a diafiltration/protein concentration device can be used, such as a centrifugal device with a porous UF membrane sized to retain the proteins, but allow the buffer to pass through. By controlling the spin time and selecting an appropriate device design, the final concentration can be controlled. For the buffer exchange to be effective, the buffer exchange step needs to be repeated two or three times (like was done with the wash and elution steps). These devices take 30-45 minutes and require multiple spins in a centrifuge. In the Amicon Ultra device commercially available from EMD Millipore, there are 5 pipette steps for buffer exchange and concentration.
As the volumes of protein samples become smaller, the undesirable potential losses of samples due to the hold-up volume within a device have become more important than ever. Current data suggest that 10 μL loss in a concentrated sample of 50 μL represents 80% protein recovery. If the protein loss were reduced by one order of magnitude from 10 μL to 1 μm, protein sample recoveries could be increased from 80% to 98%. An 18% improvement in protein sample recovery could be very valuable.
It would be desirable to provide a device and method that efficiently and effectively performs a bind and wash, a buffer exchange and concentration, and/or a complete bind, wash and elute, buffer exchange and concentration in a single device without the need to pipette transfer the precious sample between devices, particularly for sample sizes up to about 11 mL.